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  • JWH 015 synthesis The regulation of gene expression by

    2023-01-24

    The regulation of gene expression by glucocorticoids can be mediated either by the canonical mechanism that involves the interaction of the glucocorticoid receptor (GR), a transcription factor activated by the hormone, with glucocorticoid response element (Ye et al., 2007), or by the non-canonical mechanism that involves the interaction of GR with other transcription factors, in particular nuclear factor-κB (NF- κB) and activator protein-1 (AP-1) complex (Kassel and Herrlich, 2007). Both NF-κB and AP-1 are known to play an important role in gene regulation during inflammatory reaction and to regulate the transcription of inflammatory cytokines, MMPs and cyclo‑oxygenase-2 (COX-2) (Crofford et al., 1997, Bondeson et al., 2000). AP-1 is a dimeric transcription factor comprising of proteins derived from two super families the Jun (c-Jun, Jun-B and Jun-D) and the Fos (c-Fos, Fos-B, Fra-1 and Fra-2) proteins and is an important regulator of gene expression. The main AP-1 proteins in mammalian JWH 015 synthesis are Fos and Jun, which form hetero- (Jun–Fos or Jun-Fra) or homo dimers (Jun– Jun) complex (Angel and Karin, 1991, Shaulian and Karin, 2001). The number of combination of Jun and Fos creates an enormous functional diversity with individual AP-1 proteins probably engaged in different cell functions (Hess et al., 2004). Moreover, protein components of the AP-1 complex are directly involved in protein–protein interactions between various transcriptions factors (Turpaev, 2006). The AP-1 complex is a ‘signal converter’ (Wisdom, 1999), which mediates responses to cellular signals by binding DNA and producing changes in gene transcription that ultimately lead to physiologic changes in the cell. Various Fos and Jun proteins interact with the promoters of cytokine genes either individually as AP-1 dimers, or in cooperation with other transcription factors such as NF-κB, NFAT, CREB/activating transcription factor, etc. (Mechta-Grigoriou et al., 2001, Chang et al., 2002). Studies show that, the regulation of IL-4, IL-5, and GM-CSF requires the formation of NFAT/AP-1 complexes in T cells (Lee et al., 1995, Rooney et al., 1995). Similarly, in LPS-stimulated THP-1 cells, c-Jun-containing complexes have been shown to interact with NF-κB proteins p50/p65, and synergistically enhance the TNF-α promoter activity (Yao et al., 1997). More recently an increase in c-Fos expression and possibly AP-1 activity was described in human nasal polyps in vivo (Baraniuk et al., 1998). Hence the inhibition of AP-1 is a promising approach for the treatment of inflammation. In most of the cells, dexamethasone has been shown to inhibit NF-κB and AP-1 DNA binding activity (Mukaida et al., 1994, Matsumura et al., 2001). Studies reported that dexamethasone inhibits IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-κB transcription factors (Ma et al., 2004). Although literatures are available that dexamethasone reduces inflammatory cytokines via AP-1 mechanism but, which families of AP-1 factors are involved in anti-inflammatory activity of dexamethasone has not been done in lung epithelial cells. Type II alveolar epithelial cells (AECII) are since long recognized as important players of the innate immune system, producing the cytokines and chemokines (Corbière et al., 2011) which represents an important site of glucocorticoid action. Hence in the present work dexamethasone an agonist of glucocorticoid was used to study their anti-inflammatory activity towards PMA/LPS induced expression of inflammatory mediators and AP-1 factors using A549 cells. The study provides insights into the role of molecular mechanism involved in the anti-inflammatory activity of dexamethasone, and may help in identification of a target for therapeutic intervention.
    Materials and methods Human lung adenocarcinoma A549 cells were purchased from NCCS (Pune, India), Phorbol 12-myristate 13-acetate (PMA), LPS (Escherichia coli serotype O55:B5), Dexamethasone, Griess reagent, TRIzol, Oligos forward and reverse primers for different cytokines, AP-1 factors, Cyclooxygenase-2 and β-actin were designed (Patil et al., 2015), and purchased from Sigma-Aldrich (St Louis, USA). Roswell Park Memorial Institute 1640 (RPMI 1640) medium, Fetal bovine serum (FBS), penicillin, streptomycin, glutamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), trypan blue, agarose and Ethidium bromide were purchased from Himedia (Mumbai, India). Superscript reverse transcriptase for semi-quantitative RT-PCR was from Invitrogen (CA, USA). Taq DNA polymerase (1U/μl) was procured from Merck-Millipore (Mumbai, India).